Adhesion and motility are fundamental processes that gate cell metastasis. Moderate over-expression (10-30% above endogenous levels) of the adhesion plaque protein, vinculin, inhibits cell motility and increased cell adhesion, whereas down-regulation of vinculin increase cell motility and inhibits adhesion. The long-term goal of this research is to understand how vinculin regulates cell adhesion and motility. The hypothesis to tested is that the interactions of vinculin's head (Vh) and tail domain (Vt) are mechanistically important to the role of vinculin in regulating cell adhesion and motility in living cells. A tetracycline-regulated expression vector will be used to over-express 6xHisEGFP-tagged vinculin and functional mutants of vinculin in Chinese hamster ovary (CHO) cells. The relationship between the intracellular concentration of vinculin and inhibition of fibronectin (FN)- mediated cell motility (maximum migration speed at the optimal receptor occupancy), increased adhesion (mean detachment strength as a function of receptor occupancy), and rate and extent of cell spreading will be established. The effects of vinculin over-expression on stabilization of actin filaments, molecular composition of focal adhesion plaques, dynamics of adhesion plaques, filopodia, and lamellipodia, localization of vinculin, and vinculin's association with cellular proteins will be assayed by cytological analysis of fixed and living cells and by biochemical methods. The contribution of vinculin's ligand-binding activities to the over-expression phenotype will be probed through site-directed mutagenesis to knock-out individual activities. Mutants in the actin, acidic phospholipid, and Vh binding functions of Vt will be made by systematically changing all clustered charges in a window of 5 amino acids to alanine (20 mutations). Mutants will be characterize to identify Vt mutants that retain "native" structure but lose at least one of the 3 measured functions of Vt. Mutations in the VASP- and talin- binding functions will be based on known point mutations that knock out these activities in vitro. Mutations will be expressed in CHO cells in the context of individual vinculin subdomains and in the context of intact vinculin. The properties of these cells in the cell adhesion and locomotion assays will be assayed as a function of the amount of over- expressed mutant protein, and the phenotypes will be analyzed cytologically and biochemically as described, to connect phenotype with a particular ligand-binding function. New therapeutic targets for control of metastasis may be defined by this basic research on motility and adhesion.